Histochemical markers for the identification of human, monkey, guinea pig and rat tracheobronchial mucus-producing cells have been established. Stains used include Dane's, PAS-Alcian blue, mucicarmine and toluidine blue. Dissociation of tissue using 1% pronase gives a high yield of cells with a viability of greater than 90%. Attempts to purify mucus-producing cells from enzymatically-dispersed tissue using isopycnic methods have been made. Gradient materials used have included Percoll, Ficoll and bovine serum albumin. No substantial purification of this cell type has been accomplished using this approach. For this reason, we are currently using methods which separate cells on the basis of size, for example, velocity sedimentation. Epithelial outgrowths from tracheobronchial explants have been observed; however, these cells do not have the histochemical marker characteristics of mucus-producing cells. Vitamin A derivatives, feeder layers of irradiated fibroblasts, purified collagen gels and epidermal growth factor have been used in an attempt to promote functional differentiation of these cells.